Lipoprotein lipase suppression by endotoxin-induced mediator (shock assay)

ABSTRACT

A mediator substance exhibiting inhibitory effect upon anabolic enzyme activity in mammals, is prepared by a method comprising gathering a sample of macrophage cells from a mammal, incubating a portion of the macrophage cells with a stimulating mate 
     This invention was made in the course of a grant from the National Institutes of Health.

This invention was made in the course of a grant from the NationalInstitutes of Health.

RELATED APPLICATIONS

The instant application is a division of Ser. No. 414,098, filed Sept.7, 1982, which is in turn continuation-in-part of Ser. No. 351,290, nowabandoned, filed Feb. 22, 1982, by the same applicants, which in turn isa continuation-in-part of Ser. No. 299,932, now abandoned, filed Sept.8, 1981. Applicants claim the benefit of these applications under 35U.S.C. §120.

RELATED PUBLICATIONS

The applicants are authors or coauthors of two articles directed to thesubject matter of the instant invention: (1) [applicants only] "Studiesof Endotoxin-Induced Decrease in Lipoprotein Lipase Activity", J. EXP.MED. 154 at 631-639 (September, 1981, published after Sept. 8, 1981),incorporated herein by reference; and (2) [co-authors with Phillip H.Pekala and M. Daniel Lane]: "Lipoprotein Lipase Suppression in 3T3-L1Cells by an Endotoxin-Induced Mediator from Exudate Cells", PROC. NAT'L.ACAD. SCI. 79 at 912-916 (February, 1982, published after Feb. 22,1982), also incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed generally to methods and associatedmaterials for analysis of the effect and operation of invasive stimuliupon animal hosts, and in particular, is concerned with the mechanismand magnitude of the effect that such invasive stimuli may exert uponthe activity of anabolic enzymes present in the host.

2. Description of the Prior Art

Several common physiological and biochemical derangements have been seenin various mammalian hosts responding to variety of invasive stimulisuch as bacterial, viral and protozoan infections, as well as tumors andendotoxemia. For example, these responses include fever, leukocytosis,hyperlipidemia, reduced food intake and activity, and othermodifications in muscle, white blood cell and liver metabolism.Recently, a hypertriglyceridemia in rabbits infected with a protozoanparasite, Trypanosoma brucei was reported by C. A. Rouser and A. Cerami,MOL. BIOCHEM. PARASITOL. 1 at 31-38 (1980). The reportedhypertriglyceridemia was accompanied by a marked decrease in theactivity of the enzyme lipoprotein lipase (LPL) in peripheral tissues.

LPL activity has been observed by others, and it has been noted thatthis condition has existed when the human body was in shock. See E. B.Man, et al, "The Lipids of Serum and Liver in Patients with HepaticDiseases", J. CLIN. INVEST. 24 at 623, et seq. (1945); See also John I.Gallin, et al, "Serum Lipids in Infection", N. ENGL. J. MED. 281 at1081-1086 (Nov. 13, 1969); D. Farstchi, et al., "Effects of ThreeBacterial Infections on Serum Lipids of Rabbits", J. BACTERIOL. 95 at1615, et seq. (1968); S. E. Grossberg, et al., "Hyperlipaemia FollowingViral Infection in the Chicken Embryo: A New Syndrome", NATURE (London)208 at 954, et seq. (1965); Robert L. Hersch, et al., "Hyperlipidemia,Fatty Liver and Bromsulfophthalein Retention in Rabbits InjectedIntravaneously with Bacterial Endotoxin", J. LIPID. RES. 5 at 563-568(1964); and Osamu Sakaguchi, et al., "Alterations of Lipid Metabolism inMice Injected with Endotoxins", MICROBIOL. IMMUNOL. 23 (2) at 71-85(1979); R. F. Kampschmidt, "The Activity of Partially PurifiedLeukocytic Endogeneous Mediator in Endotoxin-Resistant C3H/HeJ Mice", J.LAB. CLIN. MED. 95 at 616, et seq. (1980); and Ralph F. Kampschmidt,"Leukocytic Endogeneous Mediator", J. RET. SOC. 23 (4 ) at 287-297(1978).

While the existence of "mediators" was at least suspected, the effect,if any, that they had on general anabolic activity of energy storagecells was not known. The present applicants suspected that these"mediators" exerted a depressive effect upon the activity of certainanabolic enzymes, whose reduced activity was observed in instances wherethe host entered the condition of shock in response to invasion. Thus,the relationship of the mediator produced by endotoxin-stimulatedperitoneal mouse exudate cells, upon endotoxin-sensitive andendotoxin-insensitive mice alike, and the development through thisinvestigation, of a reagent for measuring anabolic enzyme activity, wasset forth in Ser. No. 299,932, and the further investigation of thissystem in conjunction with the 3T3 L1 "preadipocyte" model system, andthe corresponding development of methods and associated materials fordeveloping antibodies to the "mediators" as well as screening proceduresfor the identification and development of drugs capable of controllingthe activity of these "mediators" was set forth in application Ser. No.351,290. The work done to date indicates that a need exists formethodology and associated diagnostic materials, to enable furtherinvestigation of the "mediator" phenomenon to proceed, as well as toprovide practical diagnostic tools useful in the treatment of theadverse sequelae of infection and concomitant shock.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, a method forpreparing a mediator substance for use in assessing the state ofanabolic enzymes in mammals, is disclosed, which finds particularutility in the instance where the mammals are undergoing invasivestimmuli such as, viral agents, bacteria, protozoa, tumors, endotoxemiaand others. In its simplest aspect, the method comprises gathering asample of macrophage cells from a mammal and incubating a portion of themacrophage cells with a stimulator material associated with an invasiveevent. For example, the stimulator material may be endotoxin, in theinstance of endotoxemia, trypanosomes, in the instance of the abovementioned protozoan parasite Trypanosoma brucei, and others.

While the peritoneal exudate cells illustrated in our present andprevious applications exemplify sources for the macrophage cells, it isto be understood that such cells may be gathered from other than theperitoneal area, and that the present invention contemplates suchvariation within its scope.

The macrophage cells and the stimulator material are incubated asindicated, and thereafter, the macrophage cells are induced to produce amediator substance capable of supressing the activity of the anabolicenzymes. Preferably, the inducement of mediator production isaccomplished during the incubation period which may, for example, extendup to about 20 hours. The resulting medium may be appropriately treatedto recover the mediator substance, and, for example, may be centrifuged,and the supernatant containing the mediator substance drawn off, or themediator may be precipitated with a 40-60% solution of ammonium sulfate.

As mentioned earlier, the mediator substance has a broad range ofeffects, including inhibitive effects that have been observed withrespect to anabolic enzymes such as lipoprotein lipase (LPL), acetylCoenzyme A carboxylase, fatty acid synthetase, and the like. Alsoinhibitive effects have been found with red blood cell formation, as themediator substance has been found to be capable of inhibiting the growthand differentiation of erythroid committed cells, by the suppression ofa number of growth and differentiation inducers, such asdimethylsulfoxide (DMSO), hexamethylene bisacetamide, butyric acid,hypoxanthine and the like, as illustrated later on herein in specificexamples.

A further embodiment of the present invention comprises a method fordetecting various invasive stimuli by their capability of inhibiting theactivity of one or more anabolic enzymes. In this method, a plurality ofmacrophage cell samples, may be prepared and selectively innoculatedwith a number of known stimulator materials, each characteristic in itseffect upon differing anabolic actors. One of the macrophage samples maybe innoculated with material from the presumed situs of the infectivestimulus, and all samples may thereafter be incubated in accordance withthe method described above. Thereafter, testing of each of thesupernatants with the mediator substances derived from the knownstimulator materials, would provide a comparative continuum for theidentification of any invasive stimulus found present. This testingmethod may utilize the 3T3 L1 cell system, for example, in the instancewhere lipoprotein lipase (LPL) activity is utilized as a parameter.Likewise, in the instance where red cell inducers are utilized, theFriend virus-transformed erythroleukemia cells may be innoculated andthereafter observed. See Friend, C., Sher, W. Holland J. G. and Sato, G.PROC. NATL. ACAD. SCI. 68, at 378-382; Marks, P. A., Rifkind, R. A.,Terada, M., Ruben, R. C., Gazitt, Y. and Fibach, E. in ICN-UCLA Symposiaon Molecular and Cellular Biology, Vol. X. "Hematopoietic CellDifferentiation". Ed. by D. W. Golde, M. J. Kline, D. Metcalf and C. F.Fox (Academic Press, New York), pp. 25-35 (1978). Naturally, othercellular systems may be utilized in the instance where specificactivities may be appropriately observed, and the invention is notlimited to the specific cellular systems set forth herein.

The invention includes methods for detecting the presence of samples ofthe various invasive stimuli in mammals by measuring mediator substanceactivity in the mammals. Thus, a number of mediator substance may beprepared from the incubation of individual cell samples with knownstimulator materials, and these mediator samples may thereafter be usedto raise antibodies capable of specifically detecting the presence ofthe respective mediator substances. These antibodies may be prepared byknown techniques, including the well known hybridoma technique forexample, with fused mouse spleen lymphocytes and myeloma, or bydevelopment in various animals such as rabbits, goats and other mammals.The known mediator samples and their antibodies may be appropriatelylabelled and utilized to test for the presence of the mediator substancein, for example, serum, as one may measure the degree of infection, anddetermine whether infection is increasing or abating, by observing theactivity of the mediator substance therein. A variety of well knownimmunological techniques may be utilized in accordance with this aspectof the present invention, including single and double antibodytechniques, utilizing detectible labels associated with either the knownmediator substances, or their respective associated antibodies.

A further embodiment of the present invention relates to a method forpreventing the occurence of shock in mammals, comprising detecting thepresence and shock promoting activity of a mediator substance in themammal, and thereafter administering an antibody to the mediatorsubstance, in an amount effective to prevent the development of shock inthe mammal.

Also, an assay system is disclosed and may be prepared for the screeningof drugs potentially effective to inhibit the synthesis or activity ofthe mediator substance. In the former instance, the effect of the testdrug on the production of mediator by stimulated macrophages isdetermined. In the latter instance, a mediator substance may beintroduced to cellular test systems, such as the 3T3 L1 cells, and theprospective drug may then be introduced to the resulting cell cultureand the culture thereafter examined to observe any changes in mediatoractivity, either from the addition of the prospective drug alone, or theeffect of added quantities of the known mediator substance.

A number of materials, compounds and agents have already been tested todetermine their effect if any on mediator substance production andactivity. As discussed in further detail in the description, infra.,only the steroid dexamethasone exhibited any inhibitory effect, and thateffect appeared to be limited to the production of the mediatorsubstance. Further agents, drugs, etc., can however be tested in themanner such as that employed with dexamethasone, and described herein.

The preparation of the mediator substance, and the determination of theimportance of its activity, has resulted in the development of numerousavenues of diagnostic and therapeutic application. It is clear from theforegoing and following, that the detection of invasive stimuli may bemade by the identification of the mediator substance, either directly orthrough the development of antibodies useful in immunological diagnosis.Further, these same antibodies may be utilized for direct treatment bycontrol of mediator activity, to avert the development of shock inmammals, while the mediator substance may be utilized as screeningagents in an assay system for the identification of drugs, agents andother substance capable of neutralizing the adverse effects of themediator substance, and thereby providing treatment of the adversesequelae of infection.

Accordingly, it is a principal object of the present invention toprovide a method for the preparation of a mediator substance exhibitingsuppressive effects upon anabolic enzyme activity in mammals.

It is a further object of the present invention to provide a method fordetecting the presence of a mediator substance in mammals in whichinvasive stimuli such as infection are suspected to be present.

It is a further object of the present invention to provide a method andassociated assay system, for screening substances, such as drugs, agentsand the like, potentially effective in combating the adverse effects ofthe mediator substance in mammals.

It is yet further object of the present invention to provide a methodfor the treatment of mammals to control the activity of said mediatorsubstance so as to mitigate or avert the adverse consequences of theiractivity.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description, which proceeds withreference to the following illustrative drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the effect of serum from endotoxin-sensitive mice treatedwith endotoxin on adipose tissue LPL activity in endotoxin-sensitivemice. Mediator activity was observed and conclusions drawn as set forthin Example 1, paragraph E herein. The data are expressed as the mean(±SEM) of six animals for each group.

FIG. 1B shows the effect of serum from endotoxin-sensitive mice treatedwith endotoxin on adipose tissue LPL activity in endotoxin-resistantmice. The data are expressed as the mean (±SEM) of three animals foreach group.

FIG. 2 shows the effect of medium from exudate cell cultures on adiposetissue LPL in endotoxin-resistant mice. The data are presented as themean (±SEM) of four or five animals.

FIG. 3 shows the effect of conditioned medium from endotoxin-treatedmouse peritoneal exudate cells over lipoprotein lipase activity of3T3-L1 cells. Data are expressed as mean SEM (n=4).

FIG. 4 shows the effect of conditioned medium from endotoxin-treatedmouse peritoneal exudate cells on the activities of acetyl CoAcarboxylase and fatty acid synthetase in 3T3-L1 cells. Three hundred(300) μl of conditioned medium was added to cultures of 3T3-L1 cells(4.2×10⁶ cells/dish) in 6 cm dishes containing 3.5 ml of DME medium and10% fetal calf serum. After the indicated times of incubation, theenzymatic activity of acetyl CoA carboxylase (identified by the symbol "") and fatty acid synthetase (identified by the symbol "o") on adigitonin releaseable cytosolic fraction of the cells was assessed.

FIG. 5 shows the effect of mediator that suppresses the synthesis ofacetyl CoA carboxylase. At the indicated times after exposure of the3T3-L1 cells to the mediator (300 μl of conditioned medium, the cellswere pulse-labeled with 0.5 mCi of ³⁵ S-methionine for 1 hour. Cytosolicfractions were obtained by digitonin treatment of a monolayer. Aliquotsof the cytosolic fractions (2×10⁵ cpm for all determinations) wereincubated with anti-acetyl CoA carboxylase and the immunoprecipitablematerial isolated and characterized as described in Example II, infra.Panel A: Autoradiogram of a 7.5%-acrylamide-0.1% SDS gel analysis ofimmunoadsorbable protein. Lane 1--control, without exposure to mediator;Lanes 2, 3, and 4--exposure of the cells to the mediator for 3, 6 and 20hours, respectively. Panel B: Results of a densitometric scan of theautoradiogram, indicating percent of immunoadsorbable material remainingrelative to control, after exposure to the mediator.

FIG. 6 shows the effect of a mediator that suppresses the synthesis offatty acid synthetase. Experimental design is identical to thatdescribed in the legend to FIG. 5. Panel A: Autoradiogram of a7.5%-acrylamide-SDS gel analysis of immunoadsorbable fatty acidsynthetase. Lane 1--control, without exposure to mediator; Lanes 2, 3,and 4, exposure of the cells to the mediator for 3, 6 and 20 hours,respectively. Panel B: Results of a densitometric scan of theautoradiogram, indicating percent of immunoadsorbable material remainingrelative to control after exposure to the mediator.

FIG. 7 shows the effect of the mediator on ³⁵ S-methionine incorporationinto protein. 3T3-L1 cells were incubated with 300 μl of conditionedmedium from endotoxin-treated mouse peritoneal exudate cells for theappropriate period and protein pulse-labeled with 0.5 mCi of ³⁵S-methionine for 1 hour. Soluble proteins were obtained by digitonintreatment of the cells, the remainder of the monolayer was extractedwith NP-40 and a membrane protein fraction obtained. Incorporation of ³⁵S-methionine into acid precipitable material was determined as describedin Example II, infra. The incorporation of radioactivity into solubleprotein () or membrane protein () following exposure of the cells to themediator are shown for the indicated time.

FIG. 8 shows the effect of mediator on protein synthesis in thecytosolic fraction of the cells. Autoradiogram of a 7.5%-acrylamide-0.1%SDS gel analysis of ³⁵ S-methionine labeled cytosolic protein afterexposure of the cells of the mediator. 3T3-L1 cells were pulse labeledand the soluble protein was obtained by digitonin as described inExample II. Aliquots (2×10⁵ cpm) of the cytosolic fraction for each timepoint were applied to the gel and electrophoresed. Lanes 1 and 2,control without exposure to mediator; Lanes 3 and 4, 1 hour exposure tothe mediator; Lanes 5 and 6, 3 hours of exposure; Lanes 7 and 8, 6 hoursof exposure; Lanes 9 and 10, 20 hours of exposure to conditioned mediumfrom mouse peritoneal exudate cells not exposed to endotoxin; Lanes 11and 12, exposure of cells to mediator for 20 hours.

FIG. 9 shows the effect of mediator on protein synthesis in the membranefraction of the cells. Autoradiogram of a 7.5%-acrylamide-0.1% SDS gelanalysis of ³⁵ S-methionine labeled membrane protein after exposure ofthe cells to the mediator. Expreimental design was identical to thatdescribed in the legend to FIG. 8. Membrane proteins were obtained byNP-40 extraction as described in Example II. Lanes 1 and 2--control,without exposure to mediator; Lanes 3 and 4, 1 hour of exposure to themediator; Lanes 5 and 6, 3 hours of exposure; Lanes 7 and 8, 6 hours ofexposure; Lanes 9 and 10, 20 hours of exposure of the cells toconditioned medium from mouse peritoneal exudate cells not exposed toendotoxin; Lanes 11 and 12, exposure to mediator for 20 hours.

FIG. 10 shows the effect of conditioned media from mouse macrophagecultures on the cell growth and heme content in Friend cells.

Friend cells (clone DS-19) were incubated for 96 hours in the absence orin the presence of Me₂ SO (1.5 vol %). Conditioned media (80 μl/ml ofgrowth medium) from mouse peritoneal macrophage cultures stimulated ornot stimulated with endotoxin (5 μg/ml) were added at the beginning ofculture. Cell members were counted with a Cytograf model 6300 andexpressed as percent inhibition of the control cells. Cell number inuntreated control culture was 3×10⁶ cells/ml. Heme content wasdetermined fluorometrically as described previously (Sassa, S., Granick,S., Chang, C. and Kappas, A. (1975) In Erythropoiesis, ed. by K. Nakao,J. W. Fisher and F. Takaku (University of Tokyo Press, Tokyo) pp.383-396). Data are the mean of duplicate determinations. The number oftrypan blue positive cells assessed by Cytograf counting was 8-10% forall cultures.

FIG. 11 shows the dose dependent effect of the endotoxin-stimulatedmacrophage mediator on cell growth and erythroid differentiation of Me₂SO-treated Friend cells. Cells were incubated for 96 hours in thepresence of 1.5% Me₂ SO with increasing concentrations of the macrophagemediator. Assays of enzymes and intermediates were performed asdescribed in Example III, infra. Data are the mean of duplicatedeterminations.

FIG. 12 shows the effect of delayed addition of the endotoxin-stimulatedmacrophage mediator on cell growth and erythroid differentiation.

Friend cells were incubated for 96 hours without changing the medium.Me₂ SO was added at time 0 to a final concentration of 1.5 vol %, whilethe endotoxin-stimulated macrophage mediator was added at the timesindicated on the abscissa (80 μl conditioned medium per ml of growthmedium). Cell number, activities of ALA dehydratase and PBG deaminase,heme and protoporphyrin contents were assayed at the end of incubationas described in Example III, infra. Data are the mean of duplicatedeterminations.

Values for control cultures treated with Me₂ SO alone were as follows:

    ______________________________________                                        Cell number   3.0    (× 10.sup.-6 /ml)                                  ALA dehydratase                                                                             3.00   (nmol PBG/10.sup.6 cells, h)                             PBG deaminase 120    (pmol uroporphyrinogen/10.sup.6                                               cells, h)                                                Protoporphyrin                                                                              0.57   (pmol/10.sup.6 cells)                                    Heme          520    (pmol/10.sup.6 cells)                                    ______________________________________                                    

FIG. 13 shows the effect of the endotoxin-stimulated macrophage mediatoron cell growth and heme content in Friend cells treated with HMBA,butyric acid, hypoxanthine or hemin.

Cells were incubated for 96 hours without changing the medium; inducingchemicals and the endotoxin-stimulated macrophage mediator (80 ladded/ml of growth medium) time 0. Final concentrations of chemicalswere mM for HMBA, 1.3 mM for butyric acid, bmM for hypoxanthine and 0.1mM for hemin. Assays were performed as described in Example III, infra.Data are the mean of duplicate determinations.

FIG. 14 shows the effect of endotoxin-stimulated macrophage mediator onthe growth and differentiation of Friend cells growing at a constantrate.

DETAILED DESCRIPTION

As disclosed in our above referenced co-pending applications on thissubject matter, we have discovered an agent which we identify herein asa mediator substance, that is produced by mammalian cells in response tostimulation by materials we refer to herein as stimulator materials,that characteristically accompany an invasive stimulus, such asbacteria, virus, some tumors, protozoa and other toxins such asendotoxemia. We have observed that the mediator substance causes themetabolism of certain of the cells of the mammal to switch from ananabolic state to a catabolic state. In particular, the mediatorsubstance appears to suppress the activity of anabolic enzymes, such aslipoprotein lipase (LPL), and the other enzymes and inducing agentslisted earlier herein. It is theorized that these mediator substance ispart of a communications system in mammals, between the immune systemand the energy storage tissues of the body. Thus, in response to variousinvasive stimuli in mammals, such as those listed before, it istheorized that the mediator substance is produced and exert an effect onenergy storage tissue such as adipose tissue, muscle, the liver, and thelike, of the impending need for energy to combat the invasion. Moreparticularly, the mediator substance may cause these storage tissues toswitch from an anabolic to a catabolic state, to facilitate the supplyof such energy. If the invasion is of short duration, the mammal canquickly recover and replenish its energy stores; however, if theinvasion is of a chronic nature, shock generally manifested by completeenergy depletion, cachexia and death, can result.

During the initial work wherein the foregoing observations were made,the method for preparing the mediator was developed, and an illustrativepreparation is set forth initially in Example I, in paragraph D, whereinperitoneal exudate cells were appropriately cultured and thereafterincubated in the presence of the known stimulator material endotoxin.After incubation, the macrophage cells are induced to produce themediator substance. In one aspect, such inducement can occur over anextended incubation, i.e. on the order of 20 hours or more. The exactperiod for such incubation, however, may vary, and the invention is notlimited to a specific time period.

Thereafter, the mediator substance may be recovered from the cellculture and stored for later use in one or more of the ways disclosedherein. Recovery may be effected by one of numerous well knowntechniques, including centrifugation and precipitation. For example, theculture described in paragraph D of Example I, was centrifuged and thesupernatant thereafter drawn off. Alternately, the mediator may beprecipitated either with a 40-60% solution of ammonium sulfate or byadsorption onto DEAE cellulose or like exchange resins. The choice ofthe particular method for recovery of the mediator substance is withinthe skill of the art.

The invention also relates to methods for detecting the presence ofinvasive stimuli in mammalian hosts by measuring the presence andactivity of the mediator substance. As mentioned earlier, the mediatorsubstance can be used to produce antibodies to themselves in rabbits,goats, sheep, chickens or other mammals, by a variety of knowntechniques, including the hydridoma technique utilizing, for example,fused mouse spleen lymphocytes and myeloma cells. The antibody can beisolated by standard techniques and utilized as a test for the presenceof the mediator substance in the suspected mammalian hosts.

Further, the antibody or antibodies can be utilized in another speciesas though they were antigens, to raise further antibodies. Both types ofantibodies can be used to determine the presence of mediator substanceactivity in the mammalian body, particularly in human serum, so as todetermine the presence of invasive stimuli such as bacterial, viral, orprotozoan infection, or the presence of certain tumors, and to followthe course of the disease. For purposes of the following explanation,the antibody or antibodies to mediator activity, will be referred to asAb₁ the antibody or antibodies raised in another species will beidentified as Ab₂.

The presence of mediator substance activity(ies) in the serum ofpatients suspected of harboring toxic levels thereof can be ascertainedby the usual immunological procedures applicable to such determinations.A number of useful procedures are known. Three such procedures which areespecially useful utilize either mediator labeled with a detectablelabel, antibody Ab₁ labeled with a detectable label, or antibody Ab₂labeled with a detectable label. The procedures may be summarized by thefollowing equations wherein the asterisk indicates that the particle islabeled, and "Med" stands for mediator activity:

    A. Med*+Ab.sub.1 =Med*Ab.sub.1

    B. Med+Ab.sub.1 *=MedAb.sub.1 *

    C. Med+Ab.sub.1 +Ab*.sub.2 =Med Ab.sub.1 Ab.sub.2 *

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized interchangeably within thescope of the present invention. The "competitive" procedure, ProcedureA, is described in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C,the "sandwich" procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the "doubleantibody", or "DASP" procedure.

In each instance the mediator substance forms a complex with one or moreantibody(ies) and that one member of the complex is labeled with adetectable label. The fact that a complex has formed and, if desired,the amount thereof, can be determined by known methods applicable to thedetection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₁ may be raised in rabbits using a mediatoras the antigen and Ab₂ may be raised in goats using Ab₁ as an antigen.Ab₂ therefore would be an anti-rabbit antibody raised in goats. Forpurposes of this description and claims, Ab₁ will be referred to as amediator activity antibody and Ab₂ will be referred to as an antibodyreactive with a mediator activity antibody or, in the alternative, an"anti-body".

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example fluorescein, rhodamine and auramine.A preferred detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate.

The mediator composition(s) can also be labeled with a radioactiveelement or with an enzyme. The radioactive label can be detected by anyof the currently available counting procedures. The preferred isotape ¹⁴C, ¹³¹ I, ³ H, ¹²⁵ I and ³⁵ S. The enzyme label can be detected by anyof the presently utlized colorimetric spectrophotometric,fluorospectrophotometric or gasometric techniques. The enzyme isconjugated to the selected particle by reaction with bridging moleculessuch as carbodiimides, diisocyanates, glutaraldehyde and the like. Manyenzymes which can be used in these procedures are known and can beutilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galacetosidase, urease, glucose oxidase plusperoxidase, galactose oxidase plus peroxidase and acid phosphatase. U.S.Pat. Nos. 3,654,090; 3,850,752; 4,016,043; are referred to by way ofexample for their disclosure of alternate labeling material, andmaterials.

High levels of mediator activity in the mammalian body may be toxic tothe mammal and cause irreversible shock. The antibody(ies) specific to amediator is useful to treat hosts suffering from this metabolicderangement. The patient can be treated, for example, parenterally, witha shock-reducing, effective dose of the antibody to neutralize at leasta portion of the mediator. The dose will, of course, vary in accordancewith the factors well known and understood by the physician orveterinarian such as age, weight, general health of the patient and theconcentration of the mediator.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of mediator substances in a suspected host. Inaccordance with the testing techniques discussed above, one class ofsuch kits will contain at least the labeled mediator or its bindingpartner, an antibody specific thereto. Another which contain at leastAb₁ together with labeled Ab₂. Still another will contain at least Ab₁and directions, of course, depending upon the method selected, e.g.,"competitive", "sandwich", "DASP" and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, test kits may be prepared with various components to detectthe mediator substance in sera or aqueous media. A first kit may beprepared to comprise:

(a) a predetermined amount of at least one labeled immuno-chemicallyreactive component obtained by the direct or indirect attachment of amediator substance or a specific binding partner thereto to a detectablelabel;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, a diagnostic test kit for the demonstration of amammal's reaction to invasive stimuli may be prepared comprising:

(a) a known amount of one mediator substance as described above (or itsbinding partner) generally bound to a solid phase to form aimmunosorbent, or in the alternative, bound to a suitable tag,;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

Additional kits may be formulated to take advantage of numerous extantimmunological protocols and techniques, and such modifications areconsidered within the scope of the invention.

In yet another aspect of the invention, antibodies specific to theaforementioned mediator may be administered in pharmaceuticalcompositions in response to shock produced by viruses, bacteria,protozoa, etc. These pharmaceutical compositions comprise:

(a) a pharmaceutically effective amount of the antibody together with

(b) a pharmaceutically acceptable carrier. With the aid of suitableliquids, the antibodies may be used in injection preparations in theform of solutions. These compositions may then be administered to ahuman in the above manner in shock-reducing amounts to dissipate, if notovercome, the effects of the invasion/shock.

As an adjunct to the development of antibodies and their use in thetechniques described above, the present invention extends to methods oftreatment of various conditions, such as shock, cachexia etc., that arefound to exist as a result of undesirably high mediator substanceactivity in the mammalian host. In such instance, the method oftreatment may include the detection of the presence and activity of theparticular mediator substance, and the subsequent administration of theappropriate antibody or antibodies to the host, in amounts effective toneutralize the undesired mediator substance activity.

Conversely, certain adverse conditions in mammals such as obesity, mayresult from excess anabolic activity. For example, obesity may be causedby undesirably high levels of activity of the anabolic enzymeslipoprotein lipase, acetyl Coenzyme A carboxylase and fatty acidsynthetase. The invention accordingly includes a method for treatingobesity, comprising administering a mediator substance in an acceptableform, and in an amount effective to assist in restoring proper bodyweight. Administration of such treatment, however, would be under strictcontrol by a physician, and the amount, manner and frequency ofadministration of the mediator would be carefully determined andconstantly monitored.

In addition to treatment with antibodies raised by a mediatorsubstances, the present invention includes an assay system, for theexamination of potential substances, such as drugs, agents, etc. toinhibit the synthesis or activity of a mediator substance. As describedearlier, appropriate cell cultures such as the 3T3-L1 and the Friendvirus transformed erythroleukemia cells may be initially treated with aparticular mediator to inhibit the activity of a particular anabolicactor, after which the potential drug etc. may be added, and theresulting cell culture observed to determine whether changes in theactivity of the anabolic actor have taken place. While the foregoingdescription makes reference to specific cell cultures for the presentassay, it is to be understood that the invention is not limited thereto.

Certain compounds have already been screened, to determine whether ornot each inhibited mediator production and/or the effect of themediator. Compounds tested and the results of such tests are set forthin the table, below.

                  TABLE                                                           ______________________________________                                                            Mediator   Mediator                                       Entity              Production Effect                                         ______________________________________                                        Dexamethasone 10.sup.-6 M                                                                         +          -                                              Aspirin 10.sup.-3 M -          -                                              Indomethacin 10.sup.-5                                                                            -          -                                              Nalaxone 10.sup.-5 M                                                                              -          -                                              Thyroid Releasing Factor 10.sup.-7 M                                                              -          -                                              ______________________________________                                          denotes yes; - denotes no)                                              

As can be seen, only dexamethasone seems to have any effect. And evendexamethasone only has an effect on "mediator" production and, thus, isonly effective at the beginning of the process. Once the mediator hasbeen produced, the dexamethasone does not seem to have any furtherimpact.

The following examples relate to the isolation of the mediatorsubstance, and the observation of its activity, as related to certainanabolic enzymes, etc. A review of the following should lend greaterappreciation to the origins and potentials of the present invention.Naturally, however, the specific materials and techniques may vary, asexplained earlier, so that the following is presented as illustrative,but not restrictive of the present invention.

It should be noted that the terms "mediator" and "mediator substance",whether used in the singular or in the plural, are intended to refer tothe same material that is isolated from macrophage cells that have beenincubated with a stimulator material as disclosed herein, and bothsingular and plural usages of these terms, where present, should beviewed as equivalent for purposes of the present disclosure. At present,the exact composition of the mediator is unknown and, therefore, alsounknown is whether the mediator is a single material or a mixture.Accordingly, the present terminology is intended to cover the "mediator"whether it is a single material or a mixture of materials. The term"mediator activity composition" and its plural may be distinct, as,although the mediator or mediator substance would be the same, theremainder of the composition may possibly vary depending upon the degreeto which other cellular constituents, factors, etc. may be presenttherein.

EXAMPLE I Isolation of Mediator Activity Compositions

A. Mice used in Testing: Male C3H/HeN endotoxin sensitive mice (7-10 wk:18-25 g) were obtained from Charles River Breeding Laboratory(Wilmington, Mass.). Male C3H/HeJ, endotoxin-resistant mice (7-10 wk:18-25 g) were obtained from The Jackson Laboratory (Bar Harbor, Maine).Mice were fed ad libitum on Rodent Laboratory Chow (Ralston Purina Co.,St. Louis, Mo.) until they were utilized. The chow diet was removed 24hours prior to each experiment and replaced with a solution of 25%sucrose in water. The animals, once injected, were only allowed accessto water. Three to 10 C3H/HeN or C3H/HeJ mice were employed in eachexperimental group.

In conducting the various experiments, each mouse was injectedintraperitoneally with one of the following: (i) 0.04 to 100 μg ofendotoxin; (ii) 0.5 ml of serum obtained from C3H/HeN mice treated withendotoxin or saline; (iii) 1 ml of medium from cultures of peritonealexudate cells of mice incubated in the presence or absence of endotoxin.Animals were sacrificed by decapitation.

B. Assay for Serum Triglyceride Concentration and Tissue LipoproteinLipase Activity: The triglyceride concentration was measured with anenzymatic assay (Triglyceride Test Set No. 961, Hycel Inc., Houston,Tex.). Lipoprotein lipase activity was assayed by the methods ofPykalisto, et al., PROC. SOC. EXP. BIOL. MED., 148 at 297 (1975); andTaskinen, et al., DIABETOLOGIA 17 at 351 (1979), both incorporatedherein, with some modifications. Epididymal fat pads were excisedimmediately after the decapitation of each mouse. The tissues wererinsed in sterile Dulbecco's Modified Eagle medium (DME) (Gibco, GrandIsland, N.Y.) containing 2% bovine serum albumin (fraction V, ReheisChemical Company, Phoenix, Ariz.) and blotted on sterile filter paper.The tissues were minced with scissors, put into pre-weighed sterilepolypropylene culture tubes (17×100 mm, Falcon Division of Becton,Dickinson and Company, Cockeysville, Md.) containing 1 ml of DME mediumsupplemented with 2% bovine serum albumin, and 2 U of heparin to releaseLPL (Lipo-Hepin, Riker Laboratories, Inc., Northridge, Calif.). Tubeswith the tissues were sealed under 5% CO₂, balance air and incubated atroom temperature with continuous gentle shaking. Tissue weight wasdetermined by the difference of the weights of the tube before and afterthe addition of the tissue. Approximately 100-300 mg of tissue wasremoved and the activity of lipoprotein lipase released from the tissuewas determined.

The enzyme assay was carried out by the method of Nilsson-Ehle andShotz, J. LIPID. RES. 17 at 536 (1976), incorporated herein, with minormodifications. The samples were incubated at 37° C. for 90 minutes ofincubation. Each sample was assayed in duplicate. One milliunit of theenzyme activity was defined as one nanomole of free fatty acid releasedper minute. The enzyme activity released per gram of wet tissue wascompared between experimental groups and control groups of each studysince there was considerable variation of LPL activity day to day. Inorder to compare the data between experiments, the data was expressed aspercent of the average activity of the control group. The range observedin C3H/HeN mice was from 32 to 59 mU/g for adipose tissue. Values to 31of 172 mU/g for adipose tissue were observed in C3H/HeJ mice.

C. Collection of Serum for Endotoxin Treated Mice:

Blood was obtained under sterile conditions from the axillary pit ofC3H/HeN mice 2 hours after i.p. injection of endotoxin (either 2 or 100μg/mouse) in 0.1 ml of saline or saline alone. Serum was prepared withinone hour after bleeding and either used immediately or kept at 80° C.until use.

D. Preparation of Endotoxin Treated Peritoneal Exudative Cells:Peritoneal exudate cells were obtained by peritoneal lavage withpyrogen-free saline (Abbott Laboratories, North Chicago, Ill.) fromC3H/HeN mice (25-33 g). These mice were injected i.p. 6 days prior tolavage with 3 ml of sterile Brewer's thioglycollate medium (DifcoLaboratories, Detroit, Mich.) to increase cell production. Theperitoneal exudate cells obtained by this procedure consist ofapproximately 60% macrophages, 20% small lymphocytes, 15% largelymphocytes, and 5% eosinophils.

The exudate cells (2×10⁶ cells/well) were incubated in serum-freeRPMI-1640 medium (Gibco, Grand Island, N.Y.) in culture platescontaining 4.5 cm² wells at 37° C. in 5% CO₂. After 3 hours, thecultures were washed three times with the medium to remove nonadherentcells. The cells which adhered to the dish were mainly macrophages. Inthe various testing procedures, the cells were incubated in serum-freeRPMI-1640 medium in the presence or absence of endotoxin (10 μg/ml). Theculture medium was removed at 26 hours incubation and centrifuged at1000 g for 5 minutes at 4° C. The supernatant was used for testingimmediately or kept at -80° C. until required for testing. No differencein activity was noted after storage for one month under theseconditions.

The various studies and isolation procedures will now be described.

E. Mediator Activity Produced in Mice: The LPL activity from adiposetissue and the serum triglyceride concentration of endotoxin-sensitivemice which had been injected with either saline (controls or 100 μg ofendotoxin) 16 hours before sacrifice was observed. This amount ofendotoxin corresponds in this strain of mice to a dose in which half theanimals die within three days after injection. It was observed that theLPL activity of adipose tissue in the endotoxin-treated animals wasdepressed to 4.5% of the control values while the triglycerideconcentration in the serum of the endotoxin treated animals wereelevated 2.6 times that of control animals.

The fact that the lowering of LPL activity is to be attributed tomediator activity produced as a result of stimulation by endotoxin andnot to the endotoxin itself is supported by the results obtained whenthe serum from endotoxin-sensitive mice which had been treated with 100μg of endotoxin 2 hours prior to bleeding was injected into anothergroup of endotoxin-sensitive mice. For this test, the control group wasinjected with serum obtained from another group of endotoxin-sensitivemice which had been injected with pyrogen-free saline. LPL activity inepididymal fat pads were measured 16 hours later.

As further illustrated in FIG. 1A, the serum from endotoxin-treated micemarkedly suppressed LPL activity in these animals compared to theactivity in the control group of animals. Since greater than 90% ofendotoxin is known to be cleared from circulation in 15 minutes, it isclear that the observed effect on LPL activity is not due to a directeffect on any remaining endotoxin present in the serum 2 hours afterinjection. It must be caused by a humoral factor or mediator produced asa result of the endotoxin injection.

To further exclude direct endotoxin effects, serum obtained from thesensitive C3H/HeN strain of mice which had been injected 2 hourspreviously with a smaller amount (2 μg) of endotoxin was injected intoendotoxin-resistant C3H/HeJ mice. The LPL activity of adipose tissue wasmeasured 16 hours after the injection to minimize the possibility ofdirect endotoxin effect and revealed a 55-percent decrease of LPLactivity as illustrated in FIG. 1B. Since resistant animals do notrespond to this small amount of endotoxin, this observation againestablishes that a humoral mediator is involved to which the resistantmice are capable of responding.

F. Mediator Activity Produced in Mice Peritoneal Exudate Cells:Experiments were undertaken to show that exudate cells could bestimulated to produce the mediator by which endotoxin suppresses the LPLactivity of adipose tissue. Exudate cells were obtained fromendotoxin-sensitive (C3H/HeN) mice by peritoneal lavage. These cellswere incubated in vitro in the presence of 10 μg/ml or absence ofendotoxin. One ml of the media from these cell cultures was injectedinto the endotoxin-resistant strain of C3H/HeJ mice. As displayed inFIG. 2, the average LPL activity in adipose tissue of animals injectedwith medium from the exudate cells incubated with endotoxin was 32% ofthat of mice which received either medium from cell cultures withoutadded endotoxin or medium containing endotoxin but without cells. Thedifference in enzyme activity between animals treated with medium fromendotoxin treated cell cultures and those animals treated with salinealone was much greater than the other controls, suggesting that a smallamount of mediator was released by exudate cells in the absence ofendotoxin and that the small amount of endotoxin in the medium withoutcells was enough to partially lower LPL activity.

From the above, it is clear that endotoxin administration markedlysuppresses adipose tissue LPL in genetic strains of mice which aresensitive to endotoxin shock and death. This action is mediated byhumoral factor or factors which can suppress adipose tissue LPL in micenot sensitive to endotoxin shock, as well as in mice which aresensitive. Peritoneal exudate cells sensitive to endotoxin are alsocapable of producing this humoral mediator.

G. Isolation of Mediator Activity Compositions from Mouse PeritonealExudate Cells: Culture medium is collected from mouse peritoneal exudatecells cultured in RPMI-1640 growth medium exposed to 10 μg/ml ofendotoxin for 24 to 36 hours and centrifuged at 500 rpm for 10 minutesat 4° C. The supernatant is subjected to ultrafiltration through anAmicon PM-10 membrane with a 10,000-Dalton cut-off. The volume of theretentate is concentrated by filtration to approximately 7 ml, placed ona Sephacryl 300 column (1.695 cm) and eluted with phosphate-bufferedsaline (PBS) (pH 7.4) at 4 ml/hr and 4° C. The volume of each collectedfraction was 3.6 ml. The fractions were analyzed for LPL activity.Fractions eluting at 108 to 115 ml and 133 to 140 ml were found to beactive in the LPL assay. The molecular weights of the mediator activecompositions in these fractions are about 300,000 and 70,000 Daltons,respectively.

The lyphylized filtrate from the ultrafiltration is dissolved in aminimal amount of distilled water, chromatographed on a Sephadex G 50column (1.6×95 cm), and eluted with PBS (pH 7.4) at a flow rate of 6ml/hr. Fractions of 3 ml were collected and analyzed for LPL activity.The activity was located in fractions eluting at 170 to 179 ml whichcorresponds to a molecular weight to about 400 to 1,000 Daltons.

The approximate molecular weights were determined in accordance withstandard practice by comparison with proteins of known molecular weight.The standards employed were ferritin, molecular weight--440,000 Daltons;bovine serum albumin, molecular weight--68,000; carbonic anhydrase,molecular weight--30,000; and ribonuclease, molecular weight--17,000;all in Daltons. As is known to those skilled in the art, molecularweights determined by this procedure are accurate to about 20%.

Mediator activity compositions can also be isolated from mouseperitoneal exudate cells by vacuum dialysis using a Millex membrane(Millipore Corporation, Bedford, Mass.) according to the followingprocedure.

Vacuum dialysis was carried out in dialysis tubing with molecular weightcut-offs at 13,000-14,000 Daltons. Samples of conditioned mediumobtained from endotoxin-treated exudate cell cultures were placed undervacuum for 6 hours at 4° C. with a 40-percent reduction in volume.Aliquots from inside and outside the bag were assayed for mediatoractivity.

It was found that all of the activity was retained during vacuumdialysis with membranes having a 12,000-Dalton pore cutoff. The mediatorcomposition isolated by this procedure, therefore, has a molecularweight greater than 12,000 Daltons. This composition contains the twohigher molecular weight compositions previously described. The reasonthat the lowest molecular weight composition is not obtained is notclear. Possibly because it is absorbed in the Millex membrane or becausethe procedure with the Amicon filter is more rapid.

The stability of the various mediator compositions to heat was assessedby heating at 100° C. for 15 minutes. The inhibitory effect of themediators on the lipoprotein lipase was completely abolished by thistreatment.

To determine whether the mediators are intracellular constituents ofnontreated cells, exudate cells were sonicated and the extract wasassayed for mediator activity. These extracts had no measurablemediator. The mediators, therefore, are not a normal intracellularsubstance of exudate cells, but are synthesized or processed in thesecells following stimulation by endotoxin.

The fact that the mediator activity compositions are in the tissueculture medium of tissue cultures of peritoneal exudate cells make itclear that they are water-soluble.

The mediators, therefore, are capable of reducing LPL activity in themammalian body, can be isolated by standard procedures such aschromatography, dialysis and gel electrophoresis from the serum ofendotoxin-treated animals or from a cell culture of peritoneal exudatecells incubated with endotoxin.

H. Studies of 3T3-L1 Preadipocytes: The properties of the mediatorcompositions were further investigated using the well defined 3T3-L1"preadipocyte" model system, by the inventors herein and co-workers, P.Pekala and M. D. Lane. 3T3-L1 preadipocytes, originally cloned frommouse embryo fibroblasts, differentiate in monolayer culture into cellshaving the biochemical and morphological characteristics of adipocytes.During adipocyte conversion, 3T3-L1 cells exhibit a coordinate rise inthe enzyme of de novo fatty acid synthesis and triacylglycerolsynthesis. Similarly, the activity of lipoprotein lipase, another keyenzyme of lipid metabolism, rises 80-180 fold during adipose conversion.The activity of the enzyme is enhanced by the presence of insulin in themedium and appears to be similar to the lipoprotein lipase of adiposetissue.

Utilizing cells of the 3T3-L1 preadipocyte cell line, it was found thataddition of the mediator compositions, derived from mouse peritonealexudate cells exposed to endotoxin as described above, suppresses theactivity of lipoprotein lipase.

The endotoxin used in the 3T3-L1 cell culture study was obtained asdescribed above. Cell culture media and fetal calf serum were obtainedfrom Gibco Laboratories (Long Island, N.Y.). 3-isobutyl-1-methylxanthinewas from Aldrich Chemical (Milwaukee, Wis.), dexamethasone from SigmaChemical Company (St. Louis, Mo.), and insulin from Eli LillyCorporation (Arlington Heights, Ill.). Triolein was from Nu Check Prep,Inc. (Elysian, Minn.). Crystalline bovine serum albumin was fromCalbiochem-Behring Corporation (LaJolla, Calif.).

I. 3T3-L1 Cell Culture: 3T3-L1 preadipocytes were cultured as previouslydescribed [MacKall, et al., J. BIOL. CHEM. 251 at 6462 (1976), and A. K.Student, et al., J. BIOL. CHEM., 255 at 4745-4750 (1980)] in Dulbecoo'smodified Eagle's medium (DME medium) containing 10% fetal calf serum.Differentation leading to the adipocyte phenotype was induced by theStudent, et al., modification of the method of Rubin, et al., [J. BIOL.CHEM. 253 at 7570-7578 (1978)]. Two days after confluence, the mediumwas supplemented with 0.5 mM isobutyl-methylxanthine, 1 μM dexamethasoneand 10 μg of insulin per ml. Forty-eight hours later, the mediumcontaining isobutyl-methylxanthine, dexamethasone, and insulin waswithdrawn and replaced with medium containing insulin at a reducedconcentration of 50 ng per ml.

J. Effect of Mediator Compositions on 3T3-L1 Cells: One hour after theculture medium was replaced with medium containing the reducedconcentration of insulin, conditioned media from cultured exudate cellswith or without added endotoxin were added to 3T3-L1 cell cultures.Incubation of the cells with the conditioned medium was carried out forup to 20 hours. At indicated times, the amount of lipoprotein lipaseactivity was measured in three compartments: (1) the activity of themedium; (2) the activity released from the cells following incubationwith heparin (this activity represents the enzyme associated with theouter surface of the cell membrane); and (3) intracellular activity.

Following the withdrawal of the medium, the dishes were rinsed once withfresh medium and the lipoprotein lipase associated with the cellmembrane was released by incubation for one hour in DME mediumsupplemented with heparin (10 U/ml) and insulin (50 ng/ml). Afterremoving this medium, the dishes were rinsed with PBS and the cells werescraped into 1 ml of 50 mM, NH₃ /NH₄ C1 buffer, pH 8.1 containingheparin 3U/ml. The cell suspension was sonicated (on ice) for 15 secondsand centrifuged at 500×g for 5 minutes. The supernatant was assayed forlipoprotein lipase.

Lipoprotein lipase assays were performed within 30 minutes after thepreparation of each sample in duplicate by the method of Nilsson-Ehleand Shotz [J. LIPID. RES. 17 at 536-541 (1976)] with minormodifications. Briefly, 75 μl of enzyme was mixed with 25 μl ofsubstrate containing 22.7 mM[3H]-triolein (1.4 uCi per mole), 2.5 mg perml of lecithin, 40 mg per ml bovine serum albumin, 33% (V/V) human serumand 33% (V/V) glycerol in 0.27M Tris-HCl, pH 8.1, and incubated at 37°C. for 90 minutes. One milliunit of enzyme activity was defined as therelease of one nanomole of fatty acid per minute. The lipase activity inall three compartments was inhibited >90% by addition of 1M NaCland >80% by omission of serum which is the source of apolipoprotein C-IIneeded for enzymatic activity.

To test the effect of the mediator on the lipoprotein lipase activity of3T3-L1 cells, the conditioned medium obtained from mouse peritonealexudate cells cultured in the presence or absence of endotoxin, wasadded to 3T3-L1 cells in monolayer culture. After a 20-hour incubationat 37° C., lipoprotein lipase activity was assessed in threecompartments: (1) the culture medium; (2) the cell surface(heparin-releasable lipase activity) and; (3) the intracellularfraction.

As shown in FIG. 3, Cols. A & C, the addition of media containing themediator substance from endotoxin-stimulated exudate cells, markedlysuppressed the lipoprotein lipase activity in all three compartments.The enzyme activities in the medium, on the cell surface (heparinreleasable), and in the intracellular compartment were 0.1%, 6%, and18%, respectively, of that of the control cells incubated with the sameamount of fresh RPMI-1640 medium. No difference in morphology or extentof adipocyte conversion was detected between cells in the experimentaland control groups. At the beginning of the study, approximately 20% ofthe cells exhibited triglyceride accumulation in the cytoplasm; 20 hourslater, approximately 50% of both the experimental and control cells hadaccumulated triglyceride.

The medium from the culture of exudate cells not treated with endotoxinhad little effect on the lipoprotein lipase activity of 3T3-L1 cells.While the medium from untreated exudate cells elicited some inhibitionin the study shown in FIG. 3, Col. B in other similar studies, mediumprepared identically had no inhibitory effect. Endotoxin itself also hada negligible inhibitory effect on lipoprotein lipase activity when theamount added was equivalent to that which might remain in theconditioned medium from endotoxin-treated exudate cells; a 19%, 9%, and0% decrease was observed on medium, heparin-releasable and intracellularcompartments, respectively. The decrease was greater (45% in medium, 17%in heparin-releasable, and 11% in the cells) when larger amounts (4.5times) of endotoxin was employed, as shown in FIG. 3, Column D.

A possible explanation for the decreased activity of lipoprotein lipasedescribed above is a direct inhibitory effect of mediators on theenzyme. This was examined by incubating medium from 3T3-L1 cell cultureswhich contained lipoprotein lipase with conditioned medium from culturesof endotoxin-treated exudate cells. It was found that the enzymeactivity was not inhibited by the mediator compositions (103% of thecontrol) at the time of mixing, and the rate of decay of enzyme activitywas the same in the experimental group and the control group. Endotoxinalso had no effect on the activity of lipoprotein lipase. The resultsimply that the mediator compositions depress lipoprotein lipase activityin 3T3-L1 cells by inhibiting the intracellular synthesis or processingof the enzyme.

The relationship between the amount of mediator compositions andlipoprotein lipase activity of 3T3-L1 cells was examined by incubatingthe cells with increasing amounts of the conditioned medium fromendotoxin-treated exudate cells for 20 hours at 37° C. Ten μl ofconditioned media according to 1.5 ml of culture media was sufficient tocause a substantial decrease in lipoprotein lipase activity, i.e., 57%decrease in the medium, 40% decrease in the heparin-releasablecompartment, and 8% decrease in the cells. Enzyme activity was furtherdepressed by increasing the amount of mediator containing medium. When250 μl were added, a decrease of greater than 95% was observed in allthree compartments. The amount of mediator present in conditioned mediumvaried somewhat from preparation to preparation.

The rate at which lipoprotein lipase activity declines after theaddition of the mediators was also investigated. Conditioned mediumcontaining mediators was added at selected intervals, and lipoproteinlipase activity was measured. A reduction of lipase activity wasapparent as early as 30 minutes after addition of 3T-3L1 cells.Approximately half of the intracellular enzyme activity was lost after2.5 hours. After 5 hours of incubation with a mediator, a maximal effectwas observed. The amount of enzyme activity in the medium and that onthe cell surface were also observed to decrease with a similar timecourse (data not shown).

The rapid decrease in lipoprotein lipase activity might reflect acompetition with insulin since removal of insulin has been shown to leadto a rapid decline in lipoprotein lipase activity in 3T3-L1 cells.However, an attempt was made to reverse the suppressive effect of themediator by increasing the concentration of insulin in the medium wasnot successful. For this study, the effect of incubating 3T3-L1 cellswith media containing insulin at various concentrations (50 ng/ml to 50μg/ml) and mediator was assessed for lipoprotein lipase activity. It wasfound that the inhibitory effect of the mediator on enzyme activity wasnot changed with increasing insulin concentrations. Even at an insulinconcentration 1,000 greater (50 g/ml) than that of standard conditions(50 ng/ml), the inhibition was not reversed.

EXAMPLE II

Reasoning that other anabolic activities of the 3T3-L1 cells might beinhibited by the mediator, we studied two key enzymes: (1) acetyl CoAcarboxylase; and (2) fatty acid synthetase; for de novo fatty acidbiosynthesis. The following example based upon a manuscript inpreparation by the inventors herein and coworkers, P. Pekala, M. D. Laneand C. W. Angus, presents evidence that the synthesis of these enzymesare also inhibited by the addition of the macrophage mediator. Theresults implicate a larger role for the mediator(s) and point to thepresence of a communication system between immune cells and energystorage cells of mammals. Presumably, during invasion the immune cellscan function as an endocrine system and selectivity mobilize energysupplies to combat the invasion.

A. Materials: Endotoxin (lipopolysaccharide) from E. coli 0127: B8isolated by the method of Westphal, described supra, was purchased fromDifco Laboratories (Detroit, Mich.). Cell culture media and fetal calfserum were obtained from Gibco Laboratories (Grand Island, N.Y.).3-isobutyl-1-methylxanthine was from Aldrich Chemical (Milwaukee, Wis.);dexamethasone, from Sigma Chemical Company (St. Louis, Miss.); andinsulin from Eli Lilly (Indianapolis, Ind.). IgG-SORB was from theEnzyme Center, Inc., (Boston, Mass.). L-[³⁵ S]Methionine (800-1440Ci/mmol) was from Amersham,. En³ Hance was obtained from NEN, (Boston,Mass.). Antiserum to fatty acid synthetase was kindly provided by Dr.Fasal Ashmad of the Papanicolau Cancer Research Institute, Miami, Fla.

B. 3T3-L1 Cell Culture: 3T3-L1 preadipocytes were cultured as previouslydescribed, [MacKall, et al., J. BIOL. CHEM. 251 at 6462 (1976)] inDulbecco's modified Eagle's medium (DME medium) containing 10% fetalcalf serum. Differentiation leading to the adipocyte phenotype wasinduced by the Student, et al., modification (A. K. Student, et al., J.BIO. CHEM. 255 at 4745-4750 (1980)) of the method of Rubin, et al., J.BIOL. CHEM. 253 at 7570 (1978). Two days after confluence, the mediumwas supplemented with 0.5 mM isobutyl-methylxanthine, 1 μM dexamethasoneand 10 μg of insulin per ml. Forty-eight hours later, the mediumcontaining isobutyl-methylxanthine, dexamethasone, and insulin waswithdrawn and replaced with medium containing insulin at a reducedconcentration of 50 ng per ml.

C. Preparation of Peritoneal Exudative Cells and Mediator Substances:Peritoneal exudate cells were obtained by peritoneal lavage from C3H/HeNmice (25-33 g; Charles River Breeding Laboratories, Wilmington, Mass.)which had been injected intraperitoneally with sterile Brewer'sthioglycollate medium (Difco Laboratories, Detroit, Mich.; 3 ml permouse) 6 days prior to harvest. The exudate cells obtained using thisprocedure are primarily macrophages with some contaminating lymphocytes,

The cells (4×10⁵ cells per cm^(2d)) were incubated in serum-freeRPMI-1640 medium for 3 hours after which nonadherent cells were removedby washing 3 times with medium. Cells adhering to the dish wereprimarily macrophages. These cells were further incubated in serum-freeRPMI-1640 medium in the presence or absence of 10 g per ml of endotoxin.After 24 hours, the culture medium was removed and centrifuged at1,000×g for 5 minutes at 4° C. The supernatant of conditioned mediumobtained from cells exposed to endotoxin was assayed and found tocontain the mediator substance that lowers LPL in 3T3-L1 cells.

No difference in activity was noted after storage of the conditionedmedium for one month at -80° C.

D. Effect of Mediator on 3T3-L1 Cells: One hour after the culture mediumwas replaced with medium containing the reduced concentration ofinsulin, conditioned media from cultured exudate cells with or withoutadded endotoxin were added to 3T3-L1 cell cultures. Incubation of thecells with the conditioned medium was carried out for up to 20 hours.

E. Labeling of Cellular Proteins: A 6-cm plate containing induced 3T3-L1cells was washed twice with 5 ml of methionine-free medium and incubatedfor 1 hour with 2 ml of the same medium containing 0.5 mCi of L-[³⁵S]-methionine during which period the rate of [³⁵ S]-methionineincorporation into cellular protein was linear. The medium was removed,the cell monolayer washed twice with phosphate-buffered saline, ph 7.4,and the soluble cytosolic proteins released by the digitonin method ofMackall, et al., supra. The remainder of the cell monolayer containingthe membranous fraction was then scraped into 2.0 ml of 100 mM HEPESbuffer, pH 7.5, containing 0.5% of the nonionic detergent NP-40 and 1 mMphenylmethylsulfonylfluoride. After trituration in a Pasteur pipet, thesuspension was centrifuged at 10,000×g for 10 minutes at 4° C., and thesupernatant saved.

[³⁵ S]-methionine incorporation into acid insoluble material wasdetermined by adding 20 μl of digitonin or NP-40 released material to0.5 ml of ice cold 20% TCA with 25 μl of 0.5% bovine serum albumin addedas carrier. After sitting at 4° C. for 1 hour, the mixture wascentrifuged at 2,000×g for 5 minutes. The pellet was incubated in 0.5 mlof 1M NH₄ OH at 37° for 30 minutes. The protein was reprecipitated onaddition of 5.0 ml of ice cold 10% TCA and filtered on Whatman GF/Cfilters. The filters were extracted with diethyl ether and the amount ofradiolabel determined.

F. Immunoadsorption Electrophoroesis: Aliquots of the soluble [³⁵S]-methionine-labeled proteins from the soluble (digitonin released)fraction of the cell monolayer were made 1 mM in PMSF and 0.5% in NP-40detergent and then added to 5 l of either antisera specific for acetylCoA carboxylase, or fatty acid synthetase, After 2 hours at 25° C., 100μl of 10% IgG-SORB were added and the labeled enzymes isolated from themixture by the method of Student, et al., supra. Polyacrylamide-SDS gelswere run according to the method of Laemmli, and prepared forfluorography by use of En³ Hance according to the manufacturer'sinstructions.

G. Results--Effect of Mediator on Acetyl CoA Carboxylase and Fatty AcidSynthetase: To examine the effect of the mediator substance on theactivities of acetyl CoA carboxylase and fatty acid synthetase enzymes,3T3-L1 cells were exposed to conditioned medium from mouse peritonealexudate cells cultured in the presence of endotoxin. After incubation ofthe 3T3-L1 cells with the mediator for 3, 6 and 20 hours, acetyl CoAcarboxylase and fatty acid synthetase activities were determined on adigitonin released cytosolic fraction of the cells (FIG. No. 4). Theactivity of both enzymes decreased over the 20-hour period toapproximately 25% of the initial values.

To determine if the loss in activity of the two enzymes was a result ofa direct effect on protein synthesis, 3T3-L1 cells were incubated withconditioned medium from cultures of endotoxin-treated exudate cells for3, 6, and 20 hours. During the final hour of incubation, the cells wereexposed to a pulse of ³⁵ S-methionine. Following the pulse, ³⁵S-methionine labelled acetyl CoA carboxylase and fatty acid synthetasewere isolated from the digitonin releasable cytosolic fractions byimmunoadsorption. Identification was accomplished by SDS-polyacrylamidegel electrophoresis and fluorography (FIGS. No. 5A and 6A). Thedecreased incorporation of ³⁵ S-methionine into immunoadsorbable acetylCoA carboxylase and fatty acid synthetase with respect to time followingexposure to the mediator is readily observed. Densitometric scanning ofthe autoradiograms (FIGS. No. 5B and 6B) indicated that after 20 hoursof exposure to the mediator, the amount of ³⁵ S-methionine incorporatedinto fatty acid synthetase and acetyl CoA carboxylase were decreased by80% and 95% respectively. These results are consistent with the conceptthat the mediator depresses the activity of acetyl CoA carboxylase andfatty acid synthetase by interfering with the synthesis of the enzyme.

H. Effect of Mediator on Protein Synthesis in General: The observedeffect on acetyl CoA carboxylase and fatty acid synthetase could beexplained by a general inhibition of protein synthesis by the mediator.To examine this possibility, the effect of mediator on amino acidincorporation into protein was investigated. 3T3-L1 cells were incubatedfor various periods of time with conditioned medium obtained from mouseperitoneal exudate cells cultured in the presence of endotoxin. ³⁵S-methionine incorporation into soluble and membrane associated proteinwas determined after 1, 3, and 6 hours of exposure of the cells to theadded factor. When 3T3-L1 cells were exposed to conditioned medium frommouse peritoneal exudate cells that were cultured in the absence ofendotoxin, no effect on ³⁵ S-methionine in incorporation into acidinsoluble protein was observed. However, as seen in FIG. No. 7,35S-methionine incorporation into TCA precipitable material in thesoluble fraction (Digitonin releasable protein) increased approximately10% in the first 3 hours with no further change observed, while a 50%decrease was observed for label incorporation into acid insolublematerial in the membrane fraction (NP-40 solubilized protein). Analysisof ³⁵ S-methionine labeled proteins following exposure to the mediatorwas accomplished utilizing SDS-gel electrophoresis. The pattern of theautoradiogram of the soluble proteins obtained on digitonin treatmentand those solublized by NP-40 of the 3T3-L1 cells are shown in FIGS. No.8 and 9. Closer inspection of FIG. No. 8 reveals the gradualdisappearance with time following the addition of the mediator of aprotein band with a molecular weight of 220,000 Daltons, while anotherband appears at approximately 18,000. In addition to these majorchanges, another new protein appears at approximately 80,000 while asecond protein of 50,000 disappears.

Analysis of the NP-40 solubilized proteins showed similar results (FIG.No. 9). Protein bands of molecular weights of approximately 80,000 and30,000 Daltons appeared while bands of approximately 220- and 50,000disappeared.

The loss of a protein band with molecular weight 220,000 in thedigitonin releasable protein, is consistent with the loss ofimmunoadsorbable acetyl CoA carboxylase and fatty acid synthetase. Theenzymes have similar molecular weights and under the conditions of thiselectrophoresis migrate with the same Rm. At present, it is not possibleto identify the other protein bands with known enzymes or proteins.

I. Analysis. The mediator appears to decrease enzymatic activity bysuppressing the synthesis of the enzymes. The effect on proteinsynthesis appears to be quite specific as there are no grossperturbations of the protein patterns observed on the autoradiograms(FIGS. No. 8 and 9). In response to the mediator, the synthesis ofseveral proteins is inhibited or induced. It was possible byimmunoprecipitation to identify fatty acid synthetase and acetyl CoAcarboxylase (M.W. 220,000) as two proteins whose synthesis is inhibitedby the mediator. The identification of the other proteins that aremodulated by the mediator is not possible at present, althoughlipoprotein lipase is a potential candidate for the 50,000-Daltonprotein that appears. The nature of proteins that are induced inresponse to the mediator and the mechanism for the modulation ofspecific protein synthesis are deserving of further improvementinvestigations.

Whether the mediator responsible for regulating the synthesis of acetylCoA carboxylase and fatty acid synthetase is the same as the mediatorthat suppresses the activity of lipoprotein lipase is not presentlyknown. The relationship of these mediator(s) to the leukocyte factorthat has been reported to metabolize amino acids from muscle to theliver is of considerable interest since this factor also imparts acatabolic state on the tissue.

EXAMPLE III

In this series of investigations, also embodied in an unpublishedmanuscript in preparation by the inventors herein, and co-worker ShigeruSassa, we sought to determine whether the macrophage mediator(s)observed in Examples I and II exerted any effect upon red blood cellsynthesis. We reasoned that, as anemia is commonly observed in mammalsafflicted with chronic infections, and that as regeneration of the redcell mass constitutes a potential drain on energy and amino acids, thebody in response to acute invasion may interrupt erythroid developmentin similar fashion and perhaps by the same mechanism observed withrespect to the anabolic enzymes lipoprotein lipase, acetyl Coenzyme Acarboxylase and fatty acid synthetase, that effect adipocytes.

To evaluate this hypothesis, we examined the effects ofendotoxin-induced factor(s) from mouse macrophages on the cellularproliferation and differentiation of a model erythroid progenativecell--the Friend virus-transformed erythroleukemia cells (See Friend, C.et al and Marks, P. A. et al., supra.). In this model system, cells canbe induced to differentiate and form hemoglobin in response to a numberof inducers, such as dimethylsulfoxide, (Friend, C., et al supra.),hexamethylenebisacetamide (Reuben, R. C. et al, PROC. NATL. ACAD. SCI.,U.S.A., 73: 862-866), butyric acid, (Leder, A. et al (1975) Cell 5:319-322), and hypoxanthine (Gusella, J. F. (1976) Cell 8: 263-269). Thisexample presents evidence that a macrophage mediator(s) can inhibit thegrowth and differentiation of erythroid committed cells, but has lesseffect on uncomitted stem cells and practically no effect on fullydifferentiated erythroid cells.

A. Materials: Endotoxin (lipopolysaccharide) from E. coli 0127: B8isolated by the method of Westpal (described supra.), was purchased fromDifco (Detroit, Mich.). A modified F12 medium was prepared in ourlaboratory (Sassa, S. et al, J. BIOL. CHEM. 252: 2428-2436 (1977)).Fetal bovine serum was purchased from GIBCO (Grand Island, N.Y.).Dimethylsulfoxide (Me₂ SO) was a product of Eastman Organic Chemicals(Rochester, N.Y.). Butyric acid and hypoxanthine were obtained fromSigma Chemical Company (St. Louis, Mo.). Hexamethylenebisacetamide(HMBA) was kindly provided by Dr. R. C. Reuben, Merck Sharp & DohmeResearch Laboratories (Rahway, N.J.).

B. Cell Culture: Murine Friend-virus transformed erythroleukemia cells(clone DS-19) were cultivated in modified F12 medium supplemented with10% heat inactivated fetal bovine serum as described previously (Sassa,S., Granick, J. L., Eisen, H. and Ostertag, W. (1978) In In vitroAspects of Erythropoiesis, ed. by Murphy, M. J. Jr. (Springer-Verlag,N.Y.) pp. 268-270).

C. Preparation of the Endotoxin-Stimulated Conditioned Medium From theCulture of Mouse Exudative Cells: Isolation of peritoneal exudate cellsfrom NCS mice (25-33 g from the Rockefeller University Breeding Colony)and preparation in vitro of an endotoxin-stimulated conditioned mediumwere carried out as described (in Example I, above). Briefly, peritonealexudate cells were isolated from mice treated with sterile Brewer'sthioglycollate medium obtained from Difco Laboratories (Detroit, Mich.),in an amount of 3 ml per mouse, 6 days prior to harvest. The cells wereincubated in serum-free RPM1-1640 medium for 3 hours, after whichnon-adherent cells were rinsed off by washing three times with medium.Cells adhering to the dish were primarily macrophages (Kawakami et al.,PROC. NATL. ACAD. SCI., USA 79:912-916; Edelson, P. S. et al., J. EXP.MED., 142: 1150-1164 (1975)).

These cells were further incubated in the serum-free medium in thepresence of endotoxin (5 μg/ml) for 24 hours. After incubation, theculture medium was removed and centrifuged at 1000×g for 5 minutes at 4°C. The supernatant of the conditioned medium contained anendotoxin-induced mediator which decreased the activity of lipoproteinlipase in 3T3-L1 cells (as reported in Example I, above) and was usedwithout further treatment.

D. Induction of Erythroid Differentiation: Two types of incubationprotocols were used to assess erythroid differentiation of Friend cells.In certain experiments illustrated in FIGS. 10-13, the cells (5×10⁴cells/ml) were incubated at 37° C., in 5% CO₂ in humidified air for 18hours. The inducing chemicals, e.g. Me₂ SO, HMBA, butyric acid,hypoxanthine or hemin were added with or without macrophage mediator(s)and cultures were incubated for 96 hours without changing the growthmedium. In other experiments such as those with results illustrated inFIG. 14, the cells (10⁵ cells/ml) were incubated for 18 hours, then Me₂SO and the macrophage mediator were added as above. The cultures weremaintained at 2×10⁵ cells/ml by diluting the cell suspension daily withfresh medium containing the chemical inducer with or without themacrophage mediator. This procedure required more macrophage mediatorthan the first experimental procedure, but made it possible to examinethe effect of mediator on rate of cell growth while cells were growinglogarithmically at a constant rate (Chang, C. S. et al; J. BIOL. CHEM.257: 3650-3654 (1982)).

E. Determination of Heme Content and Assays on the Activities of EnzymesIn the Heme Biosynthetic Pathway: The concentration of heme in cells wasdetermined by a fluorometric assay of prophyrin derivatives after theremoval of iron (Sassa, S., Granick, S., Chang, C. and Kappas, A., InErythropoisis, ed. by K. Nakao, J. W. Fisher and F. Takaku (Universityof Tokyo Press, Tokyo, Japan (1975) pp. 383-396). Cells containinghemoglobin were stained with benzidine and counted using a Cytografmodel 6300A (Sassa, S., Granick, J. L., Eisen, H., and Ostertag, W.,Supra.). Assays of aminolevulinic acid (ALA) dehydratase andporphobilinogen (PBG) deaminase were carried out by methods describedpreviously (Sassa, S., Granick, J. H., Eisen, H., and Ostertag, W.,Supra.).

F. Effects of the Macrophage Mediator on the Growth and Differentiationof Uninduced Friend Cells: Conditioned media from macrophage culturesincubated with or without endotoxin inhibited the growth of untreatedFriends cells by approximately 35% (FIG. 10, Part A.). When these cellswere incubated simultaneously with 1.5% Me₂ SO, control conditionedmedium which had not been exposed to endotoxin inhibited the cell growthby ˜42% while endotoxin-stimulated conditioned medium inhibited thegrowth of ˜60% (FIG. 10, Part B).

Heme content in these cells treated with endotoxin-stimulated ornon-stimulated conditioned media was not appreciably different from thatfound in untreated cells, indicating that the conditioned medium byitself does not affect the erythroid differentiation of Friend cells(FIG. 10, Part B). In contrast, incubation of cells with Me₂ SO andendotoxin-stimulated conditioned medium led to a significant decrease(˜40%) in the heme content in the cell (FIG. 10, Part B).

G. Dose Dependent Inhibition of Cell Growth and Differentiation By theMacrophage Mediator: When Friend cells were incubated simultaneouslywith 1.5% Me₂ SO and the endotoxin-stimulated macrophage mediator, therate of cell growth was progressively inhibited when increasing amountsof the mediator were added to the culture (FIG. 11, Part A). Aninhibitory effect of the mediator on cell growth could be detected atthe lowest concentration examined (1.12 vol. % added to growth medium).At the highest concentration (8 vol. %), the mediator inhibited cellgrowth by ˜60% compared with that of the control Me₂ SO-treated culture(FIG. 11, Part A). The decrease in cell number was not due to cell deathsince the number of dead cells as accessed by the Trypan Blue exclusiontest (Paul J. In Cell Culture) was similar (˜8%) for untreated controlsand cultures treated with the stimulated conditioned medium. Endotoxinitself (up to 15 μg/ml) exhibited no inhibitory effect on the growth ofFriend cells either in the presence or in the absence of Me₂ SO (datanot shown). These findings indicate that the endotoxin-stimulatedmacrophage mediator interferes with the growth of Me₂ SO-treated cellsmore than that of untreated cells and suggest that erythroid committedcells may be more sensitive than uncommitted stem cells to the action ofthe stimulated macrophage mediator.

Treatment of cells with the endotoxin-stimulated macrophage mediatorinhibited Me₂ So-mediated erythroid differentiation resulting in aprogressive decrease in the content of porphyrin and heme in the treatedcells as the amount of the mediator increased (FIG. 11, Part B). Theenzymatic activities of ALA dehydratase and PBG deaminase were alsodecreased by the mediator treatment (FIG. 11, Part B). The addition ofthe macrophage mediator directly to the enzyme assay mixture did notinhibit the activity of ALA dehydratase or PBG Deaminase (data notshown), ruling out a direct inhibitory effect on the activities of theenzymes.

H. Delayed Addition of the Endotoxin-Stimulated Macrophage Mediator onErythroid Differentiator: When the endotoxin-stimulated conditionedmedium was added to Me₂ SO-treated cultures at various times, it wasfound that the effect of the macrophage mediator on cell growth wasgradually lost (FIG. 12).

The effect of the macrophage mediator on erythroid differentiationdecreased more rapidly than the effect on cell growth. For example, theaddition of the endotoxin-stimulated macrophage mediator inhibited hemeand protoporphyrin formation by ˜40% at the beginning of incubation,˜25% when added at 24 hours, and had no effect when added at 48 hours orafter. Inhibition of the activity of ALA dehydratase and PBG deaminaseby the macrophage mediator treatment was also progressively diminishedwhen the mediator was added later during incubation (FIG. 12).

These findings indicate that, in contrast to the macrophage-mediatordependent inhibition of cell growth and differentiation observed inerythroid-committed cells, cells which have fully expressed erythroidcharacteristics such as those exhibiting maximal increases in theactivities of ALA dehydratase and PBG deaminase, or in the contents ofprotoporphyrin and heme, are considerably less sensitive to theinhibitory effect of the macrophage mediator.

I. Effects of The Endotoxin-Stimulated Macrophage Mediator On ErythroidDifferentiation of Friend Cells Induced by HMBA, Butyric Acid,Hypoxanthine or Hemin: In order to examine whether or not the inhibitoryeffect of the endotoxin-stimulated macrophage mediator on erythroidcommitted cells is specific for Me₂ SO-induced differentiation, weexamined the effect of the macrophage mediator on cells which wereincubated with either HMBA, butyric acid, hypoxanthine, or hemin. Wefound that the endotoxin-stimulated macrophage mediator markedlyinhibited the growth of cells incubated with HMBA, butyric acid orhypoxanthine, but not the growth of hemin-treated cells (FIG. 13, PartA). Similarly, the endotoxin-stimulated mediator inhibited the erythroiddifferentiation induced by HMBA, butyric acid or hypoxanthine, but notthat induced by hemin treatment (FIG. 13, Part B).

These findings suggest that the inhibitory action of theendotoxin-stimulated macrophage mediator on the growth oferythroid-committed cells and erythroid differentiation induced by mostof the chemical agents as represented by Me₂ SO, HMBA, butyric acid orhypoxanthine is similar, but that erythroid differentiation induced byhemin treatment is distinct in nature and not sensitive to the effect ofthe macrophage mediator. In fact the growth inhibition of Me₂ SO-treatedcells produced by the macrophage mediator alone (35%, FIG. 10) wascompletely overcome by hemin treatment (FIG. 13).

J. Effect of Endotoxin-Stimulated Macrophage Mediator on the Growth andDifferentiation of Friend Cells Growing at a Constant Rate: In order toexamine the effect of the macrophage mediator on the growth of Friendcells while they are growing at a constant rate, cells were diluted withfresh medium with or without the mediator every 24 hours to reduce thecell density to 2×10⁵ cells/ml.

Under these conditions of culture, the cells maintain a continuouslogarithmic growth at a constant rate (Chang,). C. S. et al supra.) Thetotal number of cells that would have formed from the original untreatedcontrol culture was 82×10⁶ cells/ml after 96 hours of incubation (FIG.14). The addition of the macrophage mediator significantly inhibited(˜70%) cell growth. The addition of Me₂ SO to the cultures yielded42×10⁶ cells/ml. This decrease probably reflects the growth cessationwhich is associated with terminal erythroid differentiation of thesecells. (Chang, C. S. supra.; Lo, S.C., Aft., R. and Mueller, G. C.,Cancer Res. 41: 864-870 (1981)). Combined addition of Me₂ SO and themacrophage mediator produced the most profound growth inhibition (˜90%)of these cells. Heme content in cells treated with the mediator alonewas not appreciably affected while the combined treatment with themediator and Me₂ SO brought about ˜40% inhibition of heme formation.

K. Analysis: The mediator substance under study appears to potentlyinhibit the growth and erythroid differentiation of mouse Friend-virustransformed cells. Conditioned medium from cultures not exposed toendotoxin had some inhibitory effects, but the effect of theendotoxin-stimulated conditioned medium is significantly greater ininhibiting the growth and differentiation of Friend cells. Endotoxinitself had no effect on either cell growth or differentiation.

Further, the effect of the mediator appears to be specific to certainstages of erythroid progenitor cells, in that the macrophage mediatorinhibited the growth and erythroid differentiation of uncommitted stemcells more than that of erythroid committed cells which were induced bytreatment with Me₂ SO, HMBA, butyric acid or hypoxanthine. Theinhibitory effect of the macrophage mediator on cell growth was morepronounced in cells growing logarithmically at a constant rate. Hemintreatment of Friend cells is known to cause erythroid cells maturationleading to the appearance of hemoglobinized cells but withoutaccompanying the commitment of undifferentiated stem cells to theerythroid precursor cells (Gusella, J. F., Weil, S. C., Tsiftsoglon, A.S., Volloch, V., Neuman, J. R. and Housman, D. (1976) Blood 56:481-487). Interestingly, the endotoxin-stimulated macrophage mediatoralso had very little effect on the growth and differentiation of Friendcells in the presence of hemin.

These results indicate that the endotoxin-stimulated macrophage mediatorexerts its inhibitory effect on the growth and differentiation of cellsof erythroid precursor cells including those which have been committedto undergo erythroid differentiation. On the other hand, cells whichhave fully expressed characteristics of erythroid cells such asincreased activities of ALA dehydratase and PBG deaminase, and increasedcontents of protoporphyrin and heme are no longer sensitive to theinhibitory effect of the conditioned medium. Thus it appears that theaction of the endotoxin-stimulated conditioned medium is relativelyspecific to certain early stages of erythroid precursor cells but not tofully differentiated erythroid cells.

We have also attempted to purify the mediator from theendotoxin-stimulated macrophage conditioned medium and found that ahighly purified mediator retained the inhibitory property both onlipoprotein lipase activity in 3T3 cells and on the growth anddifferentiation of Friend cells.

The macrophage factor described in this Example is believed to play arole in the pathogenesis of the anemia associated with endotoxemia orother chronic disease states, e.g., cancer, rheumatoid arthritis, wherethe activity of the reticuloendothelial system is stimulated. The Friendcell system described here should be useful to detect such in vivomediators and to elucidate the biochemical basis for the cellular effectof the mediator(s). This assay system should also aid the isolation ofthis factor and the identification of its relationship with other immunecell factors which are produced in response to invasion.

We claim:
 1. A pharmaceutical composition for the treatment of obesityin humans, comprising:A. a proteinaceous mediator substance capable ofsuppressing the activity of the anabolic enzymes lipoprotein lipase,acetyl Coenzyme A carboxylase and fatty acid synthetase, and inhibitingthe growth and differentiation of erythroid-committed cells, or aspecific binding partner thereto, wherein said mediator substanceexhibits molecular weight levels of about 300,000, 70,000 and in therange of 400 to 1000 Daltons as determined by gel electrophoresis, andis derived from animal macrophage cells that have been incubated with astimulator material that accompanies an invasive stimulus; and B. apharmaceutically effective carrier.
 2. The composition of claim 1wherein said mediator substance is further capable of inhibiting thedifferentiation of fat cells, and increasing the activity ofhormone-sensitive lipase in fat cells and the uptake of glucose inmuscle cells, while demonstrably lacking leucocyte activator activityand the ability to cause either fever or muscle protein degradation.